1. Field of the Invention
The present invention relates to a method for simultaneously surface expressing a target protein using a cofactor and an enzyme regenerating the cofactor on the cell surface.
2. Background of the Related Art
Cell surface display is a technology to express a protein or peptide fused with a proper anchoring motif on the surface of gram-negative and gram-positive bacteria, fungi, yeast, animal cells (Lee, S. Y. et al., Trends Biotechnol., 21:4552, 2003). According to the first cell surface display technology in the 1980's, a peptide or a small protein is fused with pIII, a filamentous phage, using a phage with relatively simple surface to express the peptide or protein on the surface of the phase, and thus it was called surface expression system. The cell surface display using a phage was used in screening of antibodies or screening of epitope, high-affinity ligand and the like. However, it has a problem in that the size of proteins that can be expressed on the phage surface is limited. As an alternative method, cell surface display using bacteria was developed. According to this technology, the surface protein of a microorganism such as bacteria or yeast is used as a surface anchoring motif to stably express a foreign protein on the surface of the microorganism.
In order to successively accomplish the cell surface display in microorganisms, firstly, it is necessary to select a surface anchoring motif for stably and effectively delivering the foreign protein to be expressed to the cell surface. The surface anchoring motif which has been used in E. coli so far includes membrane proteins, spore proteins, lipoproteins, autotranspoters and S-layer proteins of surface appendage. Among them, the membrane proteins are widely used as a surface anchoring motif because they have a loop structure which can pass through the cell membrane due to three dimensional structures thereof and thus can provide fusion sites for expression of various proteins.
Particularly, the cell membrane proteins such as OmpA, OmpS, LamB, OprF, PhoE and the like are used in the expression of peptides, antibodies, domains, receptors and the like, having a relatively small molecular weight (Agterberg, M. et al., gene, 88:37, 1990; Lang, H., et al., Eur. J. Bacteriol., 267:163, 2000). Since the C-terminal and N-terminal of the inserted foreign protein should be positioned stereochemically close to each other, if the protein is big, the stability of the protein is low. In practice, when a foreign protein consisting of over 50 to 60 amino acids is inserted to LamB or PhoE, a stable protein cannot be formed due to stereochemical restriction. Also, when an outer membrane porin protein of E. coli is limitedly used in the expression of, not the protein of maximum 150 amino acids but an epitope or a metal binding motif (Stahl, S. et al., Trends Biotechnol., 15:185, 1997; Kjaergaad, K. et al., Appl. Environ. Microbiol., 66:10, 2000). Recently, stable surface display of enzymes with relatively large molecular weight using membrane protein FadL, OmpC, ice nucleation protein and the like has been reported (Jung, H. C. et al., Nature Biotechnol. 16:576, 1998; Lee, S. H. et al., Appl. Environ. Microbiol. 70:5074, 2004; Lee, S. H. et al., Enzyme Microbial Technol. 35:429, 2004).
The application range of cell surface display employing secretion system of bacteria is very wide. According to the protein or peptide expressed on the surface, it can be used for various purposes. It is possible to perform the screening of peptides, antibodies, receptors and the like in a simple way by expressing a specific protein on the surface (Francisco, J. A. R. et al., Proc. Natl. Acad. USA., 91:10444, 1993), and to produce a live vaccine showing strong immune response by expressing antigen epitope on the cell surface. Also, specific enzymes needed in fine chemistry, agricultural medicines and medicines can be used as a whole cell live catalyst by expression on the cell surface and proteins which can decompose pollutants or absorb metal ions can be used in bioremediation through surface expression (Charbit, A. et al., Gene., 70:181, 1988; Sousa, C. et al., J. Bacteriol., 180:2280, 1998; Richins, R. et al., Nat. Biotechnol., 15:984, 1997).
Various enzymes are used in the process for producing substances with optical activity. Researches to produce substances with optical activity using the cell surface display system have been actively conducted. In this case, an enzyme having stereoselectivity is expressed on the cell surface and used as a whole cell catalyst (WO 2005/123924). When a hydrolase is employed in optical resolution after surface display, maximum theoretical yield is only 50%. However, unlike the hydrolase, an oxidoreductase is able to convert a prochiral substance to a substance with optical activity to have a theoretical yield of 100% and thus, attracts attention in various fields such as pharmaceutics, fine chemistry, and food and cosmetic industries (Hummel and Kula, Eur J Biochem 184:1, 1989). However, since it requires expensive materials such as NAD, NADP and FAD as a cofactor of the reaction, it is hardly applicable in the industry.
In order to solve this problem, a method comprising over-expressing an enzyme capable of regenerating a cofactor in a microorganism such as E. coli and using the microorganism as a whole cell enzyme or purifying the over-expressed enzyme and regenerating the cofactor has been studied (Yun, H. et al., Biotechnol. Prog., 21:366-371, 2005; Kula, M. R. and Kragl, U., Dehydrogenases in the synthesis of chiral compounds, in Stereoselective biocatalysis, 839-866, Ed: Patel, R. N. Marcel Dekker, Inc. New York, 2000). However, it has disadvantages in that the cofactor should pass through the microorganism to be delivered to the enzyme and thus, the efficiency of the process is low and an additional process is needed for purification of the over-expressed enzyme.
Therefore, the present inventors have found that it is possible to transform the biochemical material at a high efficiency without adding an expensive cofactor in a large amount by simultaneously surface-expressing the target protein using a cofactor and an enzyme regenerating the cofactor and using the cell in the transformation of a biochemical material. Based on the above finding, the present invention has been completed.